Eukaryotic cells ubiquitously use clathrin-mediated endocytosis (CME) to internalize nutrients, receptors and recycle plasma membrane. Defects in endocytosis are implicated in multiple diseases such as cancer, neuropathies, and metabolic syndromes and the CME machinery can be hijacked by some pathogens to infect cells. During CME, the well-conserved endocytic machinery has to overcome membrane tension to shape a ~50-nm diameter vesicle from the flat plasma membrane. When membrane tension is high, a dynamic actin cytoskeleton is necessary for CME to proceed. Despite intensive studies on most of the endocytic proteins, it remains unknown 1) how the actin network produces the forces necessary to deform the plasma membrane and 2) how its dynamic behavior adapts to different membrane tension. In this project, we will address these questions in fission yeast (S. pombe) using an innovative strategy combining experiments and mathematical modeling. We will focus our work on understanding the roles of actin assembly, the motor protein myosin-I (Myo1p), and actin filament crosslinkers fimbrin (Fim1p) and transgelin (Stg1p) during CME. In Aim 1, we will develop a 3D mathematical model taking into account individual filaments to quantitatively evaluate different molecular mechanisms for force generation and force sensing during CME, and to generate experimentally testable predictions. In Aim 2, we will use quantitative microscopy, a method we and others have developed to count locally and globally the absolute copy number of protein molecules in live cells, in order to elucidate the role of myosin-I and filament crosslinkers in force generation. In Aim 3, we will use quantitative microscopy to evaluate the molecular mechanisms by which the endocytic machinery adapts to varying membrane tension. The experimental results we will collect in aims 2 and 3 will be interpreted in light of our mathematical model developed in aim 1, which will, in turn, be continuously updated to take into account the new information and quantitative constraints we will acquire in our experiments. During this project, we will determine the roles of the actin meshwork and myosin-Is during CME and uncover new force production and mechano-sensing mechanisms at the nanometer scale that have never been characterized before.